Hematological and renal toxicity in mice after three cycles of high activity [177Lu]Lu-PSMA-617 with or without human α1-microglobulin

Radioligand therapy with [177Lu]Lu-PSMA-617 can be used to prolong life and reduce tumor burden in terminally ill castration resistant prostate cancer patients. Still, accumulation in healthy tissue limits the activity that can be administered. Therefore, fractionated therapy is used to lower toxicity. However, there might be a need to reduce toxicity even further with e.g. radioprotectors. The aim of this study was to (i). establish a preclinical mouse model with fractionated high activity therapy of three consecutive doses of 200 MBq [177Lu]Lu-PSMA-617 in which we aimed to (ii). achieve measurable hematotoxicity and nephrotoxicity and to (iii). analyze the potential protective effect of co-injecting recombinant α1-microglobulin (rA1M), a human antioxidant previously shown to have radioprotective effects. In both groups, three cycles resulted in increased albuminuria for each cycle, with large individual variation. Another marker of kidney injury, serum blood urea nitrogen (BUN), was only significantly increased compared to control animals after the third cycle. The number of white and red blood cells decreased significantly and did not reach the levels of control animals during the experiment. rA1M did reduce absorbed dose to kidney but did not show significant protection here, but future studies are warranted due to the recent clinical studies showing a significant renoprotective effect in patients.


Animals co-injected with rA1M receive lower absorbed dose to kidney
For cycle 1, only one kidney was quantified per animal, for cycle 2 one [ 177 Lu]Lu-PSMA-617 + PBS animal had both quantified, and for cycle 3, both were quantified for two [ 177 Lu]Lu-PSMA-617 + rA1M animals and two [ 177 Lu]Lu-PSMA-617 + PBS animals.Representative SPECT images can be seen in Fig. 1A and supplementary Figure S1.In the initial kidney accumulation, all animals exhibited a very quick accumulation and excretion when imaged during cycle 1, while some exhibited higher and slower accumulation during the second cycle imaging, and many more did so during the third cycle (exemplified in Fig. 1B).Supplementary Figure S2 shows the amount of decays calculated in measured kidneys for the first 48 h, note the outlier [ 177 Lu]Lu-PSMA-617 + PBS kidney in cycle 2.No statistically significant difference between groups was found for the accumulated number of decays using the Mann-Whitney U test (p-values > 0.485).The absorbed dose per injected activity value calculated for each group was applied to the injected activities and the resulting absorbed doses for each cycle can be seen in Table 1.
The post-cycle 3 imaging session at 135 days after the first injection revealed that the [ 177 Lu]Lu-PSMA-617 + PBS group had higher mean and median accumulation than [ 177 Lu]Lu-PSMA-617 + rA1M, see Fig. 2D, at both time points.This indicates a slight protection effect from rA1M, but the difference was not statistically significant (p-values > 0.099).Both PSMA groups had significantly higher accumulation than the untreated group (p-values < 0.001) using the Kruskal-Wallis test and the Bonferroni correction for multiple tests.Representative SPECT images can be seen in Fig. 2A-C.

Non-transient hematotoxicity in all [ 177 Lu]Lu-PSMA-617 treated animals
The white blood cell counts (WBC) showed a significant drop 2 weeks after the first cycle in the [ 177 Lu]Lu-PSMA-617 + PBS group that recovered after 3.5 weeks (Fig. 3A, supplementary S3-S4).The drop after the second cycle was significant in both groups compared to control mice.The WBC values did not reach the control levels again, neither after the second cycle nor after the third cycle (Fig. 3A, D).The RBC did not decrease significantly in the [ 177 Lu]Lu-PSMA-617 + PBS or [ 177 Lu]Lu-PSMA-617 + rA1M group compared to control group at either of the timepoints post-injection one (Fig. 3B, supplementary S3-S4).However, the [ 177 Lu]Lu-PSMA-617 + rA1M group decreased significantly compared to control mice after the second cycle but recovered 4 weeks after (Supplementary Figure S5-S7).The third cycle resulted in a significantly lower levels of RBC after six weeks (Fig. 3B, D) in both groups receiving [ 177 Lu]Lu-PSMA-617.The platelet values were more stable throughout the experiments for all three cycles, the only significantly lower levels were detected 4 weeks after second cycle (Fig. 3C, F, supplementary S7).All blood cell counts can be found in the supplementary materials (Supplementary Figure S3-S9).

Animals treated with [ 177 Lu]Lu-PSMA-617 had elevated albumin in urine, serum BUN and lower weight gain
To monitor the effect of each cycle on renal function, urine albumin was measured after each cycle (Fig. 4).Although no significant differences were seen between either control, [ 177 Lu]Lu-PSMA-617 + PBS and [ 177 Lu] Lu-PSMA-617 + rA1M groups, there is a clear increase of animals with albumin leakage for each cycle.The levels, however, varies greatly in both groups exposed to [ 177 Lu]Lu-PSMA-617.To account for differences in urine dilution, creatinine was measured for available samples, resulting in a significant increase of albumin/creatinine ratio in the [ 177 Lu]Lu-PSMA-617 + PBS group compared to control group (Supplementary Figure S10).Moreover, serum BUN was measured after each cycle and before sacrifice (Fig. 5).Levels of BUN was only significantly increased after the third cycle, compared to control animals.However, no difference was detected between [ 177 Lu] Lu-PSMA-617 + PBS and [ 177 Lu]Lu-PSMA-617 + rA1M groups.To further assess the kidneys, histological examination by counting viable glomeruli was performed in five random animals from each group.However, there

Discussion
Three cycles of high activity injections gave non-transient effects of radiotoxicity to the animals, measurable through increased albuminuria, a clear body weight deficit and lower numbers of circulating WBC and RBC.A significant protective effect of co-injecting rA1M could not be shown in our experimental setup, although some radioprotective effect was indicated by the lower absorbed dose.However, this model with multiple cycles of [ 177 Lu]Lu-PSMA-617 could be further used to study the resulting toxicity and strategies to diminish side effects with different interventions.We chose nude mice as our animal model, even though xenograft tumors were not implanted, mainly so our findings would be relevant for future studies that include tumors.It should be noted that nude Balb/c mice differ hematologically from their wild type counterparts, e.g. in lower WBC values 28 .At these high injected activities, the amount of injected ligand is also high and might not be comparable to studies using smaller injected amounts.Similar studies that used lower injected activities of [ 177 Lu]Lu-PSMA-617 did not find any significant difference in hematological measurements between treated animals and untreated controls, Tschan et al. at 30 MBq 29 , or between co-injection of rA1M or PBS, our previous study at 100 MBq 24 .
The absorbed doses to the kidneys calculated during this study are by necessity an approximation.The dynamic imaging immediately post injection, necessary due to variation in pharmacokinetics over time in the study, limited the number of animals per cycle, and thereby also the statistical accuracy.SPECT quantification was done by hand by a single operator, but there may still be variation between animals.Quantified accumulation per gram was used with a simulated kidney of a specific size for dosimetry since S-values are organ size-specific 30 .Both full kidneys could not always be in the field of view due to the difficulty of locating them on a low-dose CT, which could lead to over-or underestimation of accumulation in later cycles where there could be a large difference in observed effect of toxicity between kidneys in one animal.The initial kidney pharmacokinetics of [ 177 Lu]Lu-PSMA-617 were largely unchanged during dynamic scans in cycle 1 and 2 for both [ 177 Lu]Lu-PSMA-617 groups.The exception was one kidney in a [ 177 Lu]Lu-PSMA-617 + PBS animal with a drastically slower and higher accumulation in cycle 2. Initially thought to be an individual anomaly, however, we then observed the same pattern in an increased number of animals during cycle 3.Since it mathematically is an outlier value, absorbed dose calculations were done both including and excluding it.The delay in reaching maximum accumulation in the kidney, and the increase of that maximum, in later cycles seem to indicate an effect of nephrotoxicity.The post-cycle 3 imaging of [ 111 In]In-PSMA-617 had a lower kidney accumulation for the PSMA groups at 25 min post injection than was observed with [ 177 Lu]Lu-PSMA-617 during cycle 3, but this could be due to a further change in the accumulation curve.Our results show that at this level of therapy, the absorbed dose cannot be assumed from measurement during only a single cycle.
The final calculated kidney absorbed dose was lower for the group given rA1M, which might indicate a renoprotective effect for rA1M resulting from slower radioligand kinetics.However, since there were no significant differences between [ 177 Lu]Lu-PSMA-617 and [ 177 Lu]Lu-PSMA-617 + rA1M groups with regards to albumin in urine and serum BUN levels, and only four animals per group per cycle were imaged for dosimetry, this is not by itself conclusive evidence.When performing [ 111 In]In-PSMA-617 imaging post cycle 3, the measurements at 7 h p.i. show that if excluding the three kidneys from rA1M animals labelled by the analysis software, using the Tukey method 31 , as major outliers, then there would be significantly (p = 0.015) more accumulation in the [ 177 Lu]Lu-PSMA-617 + PBS group at that time, although then only 19 kidneys from 11 animals remain in the [ 177 Lu]Lu-PSMA-617 + rA1M group for analysis.The measured lower kidney absorbed dose per injected MBq for the [ 177 Lu]Lu-PSMA-617 + rA1M group in the third cycle while no significant differences between groups in the later [ 111 In]In-PSMA-617 imaging or in biomarkers, could point to either uncertainties due to low statistics in dosimetry, or a radioprotective effect of rA1M up to two cycles which was then overwhelmed by the additional absorbed dose.Future research into the potential radioprotective effect of rA1M might be wise to focus on continuous measurements of kidney function after a larger amount of individually smaller treatment cycles.Our own experience of [ 99m Tc]Tc-MAG 3 imaging after injection of 100 MBq [ 177 Lu]Lu-PSMA-617 was that it yielded small effects 24 but [ 99m Tc]Tc-DMSA imaging could be more practical and has been shown to be a good marker for kidney damage in rats after PRRT 32 .
The radiotoxic compound used in PRRT, [ 177 Lu]Lu-DOTATATE, is filtrated from the blood in the glomeruli and reabsorbed in the proximal tubule.To avoid toxic side-effects on the kidneys during PRRT, an absorbed dose limit for humans of 23 Gy for both kidneys has been adapted following experience from external radiation therapy 33 .However, the radiobiological situation in radioligand therapy is radically different than external Lu-PSMA-617 + rA1M, at both time points, the difference, however, was not statistically significant.Both PSMA groups had significantly higher accumulation than the untreated group using the Kruskal-Wallis test and the Bonferroni correction for multiple tests.★ indicates values labelled as major outliers by the statistical analysis software SPSS.***p < 0.001.radiation therapy when it comes to absorbed dose rate, spatial absorbed dose distribution etc. 17 .Therefore, the absorbed dose limit is probably higher than 23 Gy.The tolerable absorbed doses in mice might also be different than in humans.A 2015 study by Haller et al. 34 investigated kidney damage in mice after folate receptor-target RLT and found that using histological scoring, absorbed doses like those found for the [ 177 Lu] Lu-PSMA-617 + rA1M group in this study, in the 45-50 Gy range, gave moderately severe renal damage.The higher of the values for the [ 177 Lu]Lu-PSMA-617 + PBS group would fit into their results of > 69 Gy giving severe renal damage.We did not perform identical scoring but an overall histological analysis of our material, and counting of the number of glomeruli, showed less difference between [ 177 Lu]Lu-PSMA-617 mice and untreated controls than the Haller et al. one.This could be due to differences in radiotoxicity due to the microscopic distribution of the radioligand, which for folate receptor targeting is similar to [ 177 Lu]Lu-DOTATATE but might be different for [ 177 Lu]Lu-PSMA-617, something that can also be observed in that renal radiotoxicity has so far not been a clinical concern for this ligand 35 .
[ 177 Lu]Lu-PSMA-617 has specific uptake in kidney tissue via PSMA or a similar extracellular target 36,37 .The heterogeneity of the accumulation of [ 177 Lu]Lu-PSMA-617 in the kidney will affect the absorbed dose distribution and probably the nephrotoxicity but due to the comparatively low spatial resolution of the SPECT data we did not investigate this further 38 .
rA1M has protective effects in several kidney-related toxicity models, including PRRT with [ 177 Lu] Lu-DOTATATE 19,23 .It is not fully understood through which mechanism/-s rA1M act as a radioprotector although it has been suggested that rA1M utilizes its radical scavenging and reductase abilities to prevent oxidative stress-induced damage on healthy kidney tissue by reactive oxygen species resulting from radiolysis as well as dying cells 23,39 .Therefore, it is interesting that we did not detect any significant protective effects here with [ 177 Lu]Lu-PSMA-617.One reason might be a difference in renal filtration between [ 177 Lu]Lu-PSMA-617 and [ 177 Lu]Lu-DOTATATE.However, PSMA-617 is an only slightly smaller molecule than DOTATATE, 1.2 vs 1.6 kDa 40 so the filtration kinetics through the kidney should not differ substantially, and both are much smaller than A1M at 26 kDa with the recombinant forms being slightly smaller 41 .It might, however, be that the difference in pharmacokinetics and/or receptors in the kidneys would warrant another route of administration of rA1M in RLT with [ 177 Lu]Lu-PSMA-617.In Alattar et al., s.c. and i.p. administration, in addition to i.v., was evaluated and it was concluded that i.v. was preferable in the PRRT model with [ 177 Lu]Lu-DOTATATE.In humans, a continuous infusion over the course of hours might be beneficial, especially since the biological half-life in blood of A1M is quite short 18 .But to not inflict to much stress on the mice by constraining them for hours, we here instead gave them two injections (0 and 24 h p.i.), as previously described 23,24 , although it might have rendered different results with a continuous infusion instead.
Recently, rA1M was also reported to significantly reduce Major Adverse Kidney Events (MAKE) in patients undergoing cardiac surgery 42 , highlighting the clinical relevance of our study.However, it should be noted that rA1M had less effect in patients with inferior kidney function, which might be why we did not see any protective effect here; that is, the insult(s) might have damaged the animal kidneys beyond protection.
No nephrotoxicity was reported in the VISION clinical trial 10 .However, this trial was for patients with metastasized castration-resistant prostate cancer that have an overall survival measured in months post therapy.If radioligand therapy was to be administered at an earlier stage of the disease, it is possible that nephrotoxicity would become a greater concern, making radioprotection warranted 17 .
To conclude, we have shown that treating mice with three cycles of 200 MBq of [ 177 Lu]Lu-PSMA-617 gives detectable and non-transient nephrotoxicity and hematotoxicity.The high activity, multi treatment cycle design may provide a template for future studies of different types of toxicity and interventions designed to mitigate them.This study did not show a significant protective effect of rA1M but there are some indications of a preservative effect on pharmacokinetics that should be further investigated.

Radiopharmaceuticals
Radiolabeling was performed similarly to previous work 27 .Freeze-dried PSMA-617 (MedChemExpress, Monmouth Junction, NJ, USA) was dissolved in chelexed 0.2 M ammonium acetate, pH 5.5, to a final concentration of 2 µg/µL.To the reaction solution containing 60 µg (58 nmol) PSMA-617, 107 μL (3860 MBq) of no-carrier-added 177 LuCl 3 (ITM, Garching bei München, Germany) was added and the final reaction volume   www.nature.com/scientificreports/ was diluted to 350 µL using 0.2 M ammonium acetate, pH 5.5.The reaction vial was incubated at 90 °C for 60 min and then cooled at room temperature for 5 min.Thereafter 1 μL samples were taken for the analysis of the radiolabeling yield by ITLC SG (Agilent Technologies, Santa Clara, CA, USA) with 0.2 M citric acid buffer pH 2 as the mobile phase.To reduce radiolysis associated with high radioactivity concentrations, the final solution was diluted with a solution of 2% Bovine Serum Albumin (BSA) in Phosphate Buffered Saline (PBS) containing 100-fold molar excess EDTA (Ethylenediaminetetraacetic acid disodium salt dihydrate).The final volume was 100 µL (201 MBq) per injected dose.The purity of the final solution was further confirmed using instant thinlayer chromatography.Preparation of [ 111 In]In-PSMA-617 was performed under the same labeling conditions of pH and temperature.In brief, 185 MBq of 111 InCl 3 was incubated with 60 µg (58 nmol) PSMA-617 for 60 min before the mixture was allowed to cool down and 1 μL samples were analyzed using ITLC SG.The final solution was diluted with a solution of 2% BSA in PBS containing 100-fold molar excess EDTA.

In vivo studies
Male BALB/cAnNRj nude mice (Janvier Labs, Le Genest-Saint-Isle, France) were used for in vivo studies.All Animals were given ad libitum access to food and water.An overview of the in vivo studies can be found in Fig. 7.
Every 5 weeks, animals in the [ 177 Lu]Lu-PSMA-617 groups were injected i.v.first with either 5 mg/kg (approx.30 µL) rA1M (Guard Therapeutics International AB, Stockholm, Sweden, n = 12) or 30 µL PBS (n = 12) and then directly with approximately 200 MBq, 3.0 nmol, 100 µL [ 177 Lu]Lu-PSMA-617 with repeated administration of rA1M or corresponding volume of PBS again 24 h later.One group was only injected with PBS (control, n = 6).The activity of the 177 Lu syringe was measured before and after injection to record the correct injected activity.Animals were given a total of three cycles (approximately 600 MBq).Animals were weighed throughout the study and all animals were sacrificed after 142 days post first injection.
An additional group of 6 animals were given 50 MBq, 0.8 nmol, 25 µL [ 177 Lu]Lu-PSMA-617 each i.v.Three of these were sacrificed at 2 weeks p.i. and three at 3 weeks p.i. Kidneys were excised, weighed and the activity measured in a well counter (2480 Wizard 2 automatic, PerkinElmer, Waltham, MA, USA) to calculate percent of injected activity per gram.

Dosimetry and radioligand accumulation
For every cycle, 4 animals from the [ 177 Lu]Lu-PSMA-617 + rA1M group and 4 animals from the [ 177 Lu] Lu-PSMA-617 + PBS group were selected for imaging for dosimetry.No animal was selected for more than one cycle.These animals were anesthetized with 2-3% Isoflurane (Baxter, Deerfield, IL, USA) in a O 2 and N 2 O mix, placed in the animal bed of the SPECT/CT (NanoSPECT/CT Plus, Mediso; Budapest, Hungary), and immediately a CT scan was performed.The field-of-view for SPECT imaging was set to the location of the kidneys on the CT image.Injections were performed, and SPECT imaging started approximately half a minute post 177 Lu injection on a protocol of dynamic imaging with 90-100 frames of 1 min length each.A 1-h static SPECT image of the whole animal was also acquired at 7 h p.i. and at 48 h p.i.For the first cycle, an additional static SPECT image was taken at 96 h p.i. but it was calculated that this could be substituted by correcting the final mean absorbed dose per injected activity (Gy/MBq) result by a factor of 0.9019 when only using 7 and 48 h p.i. static scans, introducing only a mean error of 1.2%.Also, during the first cycle, a standard of known 177 Lu activity was included in the field-of-view to ensure that the high injected activity did not affect the detector efficiency.All SPECT images were reconstructed using HiSPECT software (SciVis, Göttingen, Germany) and the "Standard" pre-set.
SPECT images were quantitatively analysed using VivoQuant 3.0 software (inviCRO, Boston, MA, USA).Any activity measured on the static scans as remaining in the tail of the animal was subtracted from the injected activity when calculating accumulation in each image, and decay corrected from the 7 h p.i. scan to also be included for calculations of the dynamic scan.

Figure 1 .
Figure 1.Uptake characteristics of [ 177 Lu]Lu-PSMA-617 changes after each cycle of treatment.Representative SPECT images and graphs of [ 177 Lu]Lu-PSMA-617 activity in male BALB/cAnNRj nude mice.Maximum intensity projections using the NIH color scale windowed to best display kidneys with the top activity noted.Note that the Cycle 2 animal is the one with an outlier kidney.Annotations: k = kidney, and s = standard.(A) Sum of all frames of the dynamic scans immediately p.i. with 90-100 frames of 1 min.(B) Examples of [ 177 Lu] Lu-PSMA-617 kinetics in kidneys as measured using dynamic SPECT imaging directly after injection.Percent of injected activity per gram of kidney (%IA/g) plotted.Chosen animals have typical distribution for their cycles except for the second cycle animal where the high uptake kidney is an outlier.

Figure 2 .
Figure 2. Different uptake of [ 111 In]In-PSMA-617 between treated and untreated animals but not between animals co-injected with rA1M or not.SPECT images of the 111 In activity in animals convolved by a 2 mm FWHM Gauss filter, taken 7 h p.i. [ 111 In]In-PSMA-617 at 135 days post first 177 Lu injection.Maximum intensity projections using the "Hot" color scale windowed to best display kidneys with the top activity noted.Annotations: b = bladder and k = kidney.(A) Animal from the [ 177 Lu}Lu-PSMA-617 + rA1M group.(B) Animal from the [ 177 Lu}Lu-PSMA-617 + PBS group.Outlier in cycle 2. (C) Animal from the untreated control group.(D) Kidney accumulation (%IA/g) of [ 111 In]In-PSMA-617 at end of study 135 days post first injection.The [ 177 Lu]Lu-PSMA-617 + PBS group had higher mean and median accumulation than [ 177 Lu]Lu-PSMA-617 + rA1M, at both time points, the difference, however, was not statistically significant.Both PSMA groups had significantly higher accumulation than the untreated group using the Kruskal-Wallis test and the Bonferroni correction for multiple tests.★ indicates values labelled as major outliers by the statistical analysis software SPSS.***p < 0.001.

Figure 3 .
Figure 3. Animals treated with [ 177 Lu]Lu-PSMA-617 had a decrease in blood cell counts.Blood cell counts during the course of the experiment.Percent of WBC (A), RBC (B) and PLT (C) compared to control animals.Number of WBC (D), RBC (E), and PLT (F) 6 weeks after last cycle.Dotted line represents when new cycle where administered.Data are presented as line charts (A-C) or scatter plots with mean (± SEM) (D-F).Statistical Statistical comparison between groups was made with one-way ANOVA with a Tukey's multiple comparisons post hoc test (D-F).Only significant differences are presented in the figure.*p < 0.05, *** p < 0.001, **** p < 0.0001.

Figure 4 .
Figure 4. Animals treated with [ 177 Lu]Lu-PSMA-617 had elevated albumin in urine.Albumin levels in urine 2 days after first cycle (A), three weeks after first cycle (B).after second cycle (C), after third cycle (D) and over time (E).Data are presented as scatter plots with mean (± SEM).Statistical Statistical comparison between groups was made with one-way ANOVA with a Tukey's multiple comparisons post hoc test.

Figure 5 .
Figure 5. Animals treated with [ 177 Lu]Lu-PSMA-617 had elevated serum BUN levels after three cycles.BUN serum levels after first cycle (A), after second cycle (B).after third cycle (C), after third cycle before sacrifice (D).Data are presented as scatter plots with mean (± SEM).Statistical comparison between groups was made with one-way ANOVA with a Tukey's multiple comparisons post hoc test.Only significant differences are presented in the figure.*p < 0.05, **p < 0.01.

Figure 6 .
Figure 6.Animals treated with [ 177 Lu]Lu-PSMA-617 had lower weight gain.Changes in body mass (calculated as fraction body mass increase; body mass increase/baseline body mass) after 1 (A), 2 (B) and 3 cycles (C).Body mass presented as group mean (± SEM) during the experiment (D), the dotted lines represent each cycle of [ 177 Lu]Lu-PSMA-617 injected.Data are presented as scatter plots with mean (± SEM).Statistical comparison between groups (A-C) was made with Kruskal-Wallis test (Dunn's multiple comparison test).Only significant differences are presented in the figure.*p < 0.05, ** p < 0.01.

Figure 7 .
Figure 7. Scheme of in vivo experiments.Time schedule for the main treatments of the three animal groups.

Table 1 .
Animals co-injected with rA1M receive lower absorbed dose to kidney.Mean injected activity, excluding any activity extravasated in the tail at time of imaging.*Including outlier kidney in cycle 2. Absorbed dose per injected activity and resulting absorbed dose are each calculated 5 weeks p.i.Note that the values differ substantially whether an outlier kidney in cycle 2 is included or not, thus both results are included.